706_S2006_PS3key - 7.06 Problem Set #3, 2006 1. You are...

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7.06 Problem Set #3, 2006 1. You are studying the EGF/Ras/MAPK pathway in cultured cells. When the pathway is activated, cells are signaled to proliferate. You generate various mutants described below. (Assume that none of the mutations affect the ability of the proteins to fold properly.) Assume that, when you add ligand (EGF), it is present in abundance. You perform each of your studies in two ways: (i) Overexpression of the mutant while the wild-type endogenous gene is present (ii) Replacement of the endogenous gene with the mutant gene In each case, predict the outcome and explain your reasoning. Your choices are: constitutive signaling (+/- ligand), no signaling (+/- ligand), or no effect (proliferation in the presence of ligand and no proliferation in the absence of ligand). a) Mutation of the EGF receptor so that dimerization is not possible. (i) No effect. The mutant EGF receptors cannot dimerize and signal, but the wild-type receptors retain the ability to signal. (ii) No signaling. The mutant EGF receptors cannot dimerize and signal b) Mutation of the proline-rich domain of Sos to an alanine-rich domain. (i) No effect. Grb2 is unable to bind to Sos via its SH3 domain, because GRB2 binds to the proline rich domain of Sos. However, wild-type Sos can still bind to GRB2 and trigger Ras activation. (ii) No signaling. Grb2 is unable to bind to Sos via its SH3 domain, because GRB2 binds to the proline rich domain of Sos. c) Mutation of the SH2 domain of Grb2 so that it is non-functional. (i) No effect. Mutant Grb2 is unable to bind to the activated phosphorylated form of the EGF receptor, but the wild-type Grb2 retains this ability. (ii) No signaling. Mutant Grb2 is unable to bind to the activated phosphorylated form of the EGF receptor d) Mutation of the SH3 domain of Grb2 such that it is non-functional. (i) No signaling. If GRB2 has no SH3 domain, then it cannot bind and activate Sos. This would lead to the inability of the Ras pathway to signal, regardless of whether wild-type GRB2 is present. This is because this form of GRB2 is dominant negative, as it retains its ability to bind to RTKs, and thus occupies the RTK and prevents wild-type GRB2 from binding.
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(ii) No signaling. If GRB2 has no SH3 domain, then it cannot bind and activate Sos. e) A serine-to-alanine mutation at each of the serines in the N-terminal regulatory domain of Raf to which 14-3-3 binds. (i) Constitutive. This is a dominant mutant. Alanines cannot be phosphorylated, and thus 14-3-3 cannot bind and regulate the mutant Raf, as it is never phosphorylated. Thus, the mutant Raf is always active, regardless of the presence or absence of ligand. (ii) Constitutive. Alanines cannot be phosphorylated, and thus 14-3-3 cannot bind and regulate the mutant Raf, as it is never phosphorylated f) A serine-to-aspartate mutation at each of the serines in the N-terminal regulatory domain of Raf to which 14-3-3 binds. (i) No effect. The aspartate mimics phosphorylated serine.
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706_S2006_PS3key - 7.06 Problem Set #3, 2006 1. You are...

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